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BACKGROUND: In recent years, the opioid crisis has had devastating effects on communities across the country. In the wake of 4 University of Southern California (USC) student overdose deaths in 2019, USC pharmacy students initiated a naloxone distribution program called NaloxoneSC. This program has distributed free naloxone kits, fentanyl test strips, and overdose prevention training to hundreds of students thus far. This report aims to provide a framework for the implementation of similar harm reduction programs at other universities. OBJECTIVES: NaloxoneSC partnered with USC Student Health to implement a naloxone distribution program that expands access to naloxone and increases opioid overdose education within the USC community. SETTING: USC undergraduate campus and Health Sciences Campus. METHODS: Students can follow the steps for obtaining a naloxone kit listed on the NaloxoneSC website. Watching a 10-minute training video or attending a naloxone training workshop completes the opioid overdose education. The kit is picked up by the student from 1 of 2 USC Student Health Center locations, Engemann or Eric Cohen. Each kit is packaged with Narcan nasal spray and fentanyl test strips. RESULTS: In 1 year, the program received at least 320 naloxone kit requests. Demand for naloxone kits increased over time, and roughly 4 times as many students requested kits from Engemann than from Eric Cohen. Approximately 600 students have received opioid overdose education through NaloxoneSC. CONCLUSIONS: In 1 year, more than 300 USC students voluntarily requested naloxone kits from NaloxoneSC. These findings suggest a need for increased access to this resource on college campuses. Peer-to-peer education and naloxone kit distribution are feasible and effective strategies that help address public health concerns and reduce harm among university students. NaloxoneSC can serve as an example for other college campuses or entities to newly implement or expand their own harm reduction measures.
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Overdose de Drogas , Overdose de Opiáceos , Estudantes de Farmácia , Humanos , Naloxona/uso terapêutico , Antagonistas de Entorpecentes/uso terapêutico , Overdose de Opiáceos/tratamento farmacológico , Redução do Dano , Overdose de Drogas/tratamento farmacológico , Overdose de Drogas/prevenção & controle , Fentanila/uso terapêuticoRESUMO
BACKGROUND: Studies have shown a relationship between increased iron content and clinical progression, cognitive impairment, and brain atrophy in patients with multiple sclerosis. Altered phase, as determined by susceptibility-weighted imaging (SWI), can potentially capture iron content changes. OBJECTIVE: The objective of this study was to investigate phase changes in white matter (WM) lesions and subcortical deep-gray matter (SDGM) of patients with relapsing-remitting (RR) MS treated with interferon beta-1a administered subcutaneously versus untreated healthy controls (HCs). METHODS: We conducted a 24-week, nonrandomized, open-label pilot study of 23 patients with RRMS receiving interferon beta-1a administered subcutaneously and 15 HCs. Patients were imaged on a 3T scanner at baseline, 12, and 24 weeks; changes in phase behavior in WM lesions and regional SDGM [mean phase of low-phase voxels (MP-LPV)], and in SDGM volumes, were measured. Between- and within-group changes were tested using nonparametric statistics adjusted for multiple comparisons. RESULTS: The number (p = 0.003) and volume (p < 0.001) of phase WM lesions both significantly decreased among RRMS patients over 24 weeks. At baseline, MP-LPV was lower (suggestive of greater iron content) in total SDGM among RRMS patients versus HCs (p = 0.002). Week 24 MP-LPV changes from baseline were not significantly different between groups in total SDGM or any region except the putamen (-0.0025 radians in RRMS patients versus 0.0035 radians in HCs; p = 0.041). CONCLUSIONS: Over 24 weeks, phase lesions were reduced significantly in the RRMS group. These preliminary results suggest that SWI-filtered phase may become a useful tool for monitoring RRMS disease activity.
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Subanalysis of a pilot study (NCT01085318) assessed correlations between serum ferritin and imaging assessments in relapsing-remitting multiple sclerosis patients (n = 23) receiving 44 µg interferon beta-1a subcutaneously three times weekly. At baseline, 12, and 24 weeks, mean ferritin was 75, 127 (p < 0.001 vs baseline), and 101 (p=0.020 vs baseline) ng/mL. No relationship between ferritin and susceptibility-weighted imaging (SWI)-filtered phase of subcortical deep gray matter was found. Increasing ferritin correlated with decreasing lesion numbers on both fluid attenuated inversion recovery and SWI phase at 12 weeks (r = -0.62; p = 0.003; n = 21), and with decreasing gadolinium-enhancing lesion volume at 24 weeks (r = -0.71; p = 0.050; n = 8).
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Ferritinas/sangue , Interferon beta/administração & dosagem , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adulto , Biomarcadores/sangue , Suscetibilidade a Doenças , Esquema de Medicação , Feminino , Humanos , Injeções Subcutâneas , Interferon beta-1a , Masculino , Pessoa de Meia-Idade , Projetos PilotoRESUMO
Maternal-to-embryonic transition (MET) is the period in early embryonic development when maternal RNAs and proteins stored in the oocyte are gradually degraded and transcription of the embryonic genome is activated. First insights into the timing of embryonic genome activation (EGA) came from autoradiographic analyses of embryos following incorporation of [(3)H]uridine. These studies identified the eight- to 16-cell stage of bovine embryos as the period of major EGA, but detected first transcriptional activity already in one-cell embryos. Subsequent studies compared the transcriptome profiles of untreated embryos and of embryos incubated with the transcription inhibitor α-amanitin to reveal transcripts of embryonic origin. In addition, candidate gene-based and global gene expression studies over several stages of early development were performed and characteristic profiles were revealed. However, the onset of embryonic transcription was obscured by the presence of maternal transcripts and could only be determined for genes which are not expressed in oocytes. Using RNA sequencing of bovine germinal vesicle and metaphase II oocytes, and of four-cell, eight-cell, 16-cell and blastocyst stage embryos, we established the most comprehensive transcriptome data set of bovine oocyte maturation and early development. EGA was analyzed by (i) detection of embryonic transcripts which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. Using these three approaches we were able to map the onset of embryonic transcription for almost 7400 genes. Genes activated at the four-cell stage or before were functionally related to RNA processing, translation, and transport, preparing the embryo for major EGA at the eight-cell stage, when genes from a broad range of functional categories were found to be activated. These included transcriptional and translational functions as well as protein ubiquitination. The functions of the genes activated at the 16-cell stage were consistent with ongoing transcription and translation, while the genes activated in blastocysts included regulators of early lineage specification. Fine mapping of EGA provides a new layer of information for detecting disturbances of early development due to genetic, epigenetic, and environmental factors.
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Bovinos/embriologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genoma/fisiologia , Análise de Sequência de RNA/veterinária , Animais , Sequência de Bases , Perfilação da Expressão GênicaRESUMO
The association between clinical outcomes and abnormal susceptibility-weighted imaging (SWI)-filtered phase, indicative of increased iron content, as well as atrophy, was investigated in the subcortical deep-gray matter (SDGM) of multiple sclerosis (MS) patients. 149 relapsing-remitting (RR) and 61 secondary-progressive (SP) MS patients underwent SWI on a 3T scanner. Mean phase of the abnormal phase tissue (MP-APT) and normalized volumes were determined for the total and region-specific SDGM structures. In an age- and gender-adjusted regression model, total SDGM volume was the strongest predictor of Expanded Disability Status Scale (EDSS) (beta = -.224, p<.001), followed by total SDGM MP-APT (beta = -.168, p <.019). This model accounted for 30.4% of the variance in EDSS. Only SDGM MP-APT added additional variance in predicting EDSS, compared to conventional MRI metrics. Caudate and red nucleus MP-APT and amygdala volume were associated with EDSS. Our findings suggest that disability in MS patients is associated better with SDGM pathology, as indicated by increased iron content and atrophy, than with lesion burden or white matter and cortical volumes.
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Encéfalo/patologia , Ferro/metabolismo , Imageamento por Ressonância Magnética/métodos , Esclerose Múltipla/patologia , Neuroimagem/métodos , Fatores Etários , Encéfalo/metabolismo , Feminino , Humanos , Masculino , Esclerose Múltipla/metabolismo , Análise de Regressão , Fatores Sexuais , Estatísticas não ParamétricasRESUMO
OBJECTIVE: The objective of this paper is to assess abnormal phase values, indicative of increased iron content, using susceptibility-weighted imaging (SWI)-filtered phase of the subcortical deep gray matter (SDGM) in adolescent multiple sclerosis (MS) and other neurological disorders (OND) patients, and in healthy controls (HC). METHODS: Twenty adolescent MS and eight adolescent OND patients and 21 age- and sex-matched HC were scanned on a 3T GE scanner. Mean phase of abnormal phase tissue (MP-APT), MP-APT volume, normal phase tissue volume (NPTV) and normalized volume measurements were obtained for total SDGM, as well as specific structures separately. RESULTS: Significantly increased MP-APT (28.2%, p<.001) and MP-APT volume (82.7%, p<.001), and decreased NPTV (-23.3%, p<.001) and normalized volume (-15.5%, p<.001) in the pulvinar nucleus of the thalamus was found in MS patients compared to HC. MP-APT in MS patients was also increased in total SDGM (p=.012) and thalamus (p=.044). Compared to OND patients, MS patients had increased MP-APT volume in the pulvinar nucleus of the thalamus (p=.044) and caudate (p=.045). Increased MP-APT of the SDGM structures were associated with increased T2 and T1 lesion burden and brain atrophy in MS patients. CONCLUSION: Adolescent MS patients showed increased iron content in the SDGM compared to OND patients and HC.
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Ferro/metabolismo , Esclerose Múltipla Recidivante-Remitente/metabolismo , Fibras Nervosas Amielínicas/química , Pulvinar/química , Adolescente , Estudos de Casos e Controles , Criança , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Fibras Nervosas Amielínicas/metabolismo , Pulvinar/metabolismoRESUMO
PURPOSE: To investigate phase lesions identified on susceptibility-weighted imaging (SWI)-filtered phase images in patients with multiple sclerosis (MS), clinically isolated syndrome (CIS) and healthy controls (HC). To relate phase lesion characteristics to other clinical and MRI outcomes. MATERIALS AND METHODS: 95 relapsing-remitting (RR), 40 secondary-progressive (SP) MS patients, as well as 19 CIS patients and 49 age- and sex-matched HC, were scanned on a 3T scanner. Phase-, T1-, and T2-lesion characteristics were determined. Overlap of T1- and T2-weighted imaging (WI) lesions with phase lesions (T1P and T2P), as well as brain atrophy outcomes, was assessed. RESULTS: MS patients showed significantly greater numbers and larger volume of phase lesions, compared with HC (P < 0.001). 23.6% of T2 lesions overlapped with phase lesions, whereas the same figure for T1 lesions was 37.3%. Conversely, 33.4% and 69.7% of phase lesions were not visible on T2- or T1-WI, respectively. Phase, T1P and T2P lesions were not related to clinical outcomes, but phase lesions were related to ventricular enlargement. CONCLUSION: Phase lesions were present in both MS and CIS patients, and showed partial overlap with lesions observed using conventional MRI. The role of phase lesions in clinical progression remains unclear and should be further explored.
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Distúrbios do Metabolismo do Ferro/complicações , Distúrbios do Metabolismo do Ferro/patologia , Imageamento por Ressonância Magnética/métodos , Esclerose Múltipla/complicações , Esclerose Múltipla/patologia , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Information-processing speed (IPS) slowing is a primary cognitive deficit in multiple sclerosis (MS). Basal ganglia, thalamus and neocortex are thought to have a key role for efficient information-processing, yet the specific relative contribution of these structures for MS-related IPS impairment is poorly understood. To determine if basal ganglia and thalamus atrophy independently contribute to visual and auditory IPS impairment in MS, after controlling for the influence of neocortical volume, we enrolled 86 consecutive MS patients and 25 normal controls undergoing 3T brain MRI and neuropsychological testing. Using Sienax and FIRST software, neocortical and deep gray matter (DGM) volumes were calculated. Neuropsychological testing contributed measures of auditory and visual IPS using the Paced Auditory Serial Addition Test (PASAT) and the Symbol Digit Modalities Test (SDMT), respectively. MS patients exhibited significantly slower IPS relative to controls and showed reduction in neocortex, caudate, putamen, globus pallidus, thalamus and nucleus accumbens volume. SDMT and PASAT were significantly correlated with all DGM regions. These effects were mitigated by controlling for the effects of neocortical volume, but all DGM volumes remained significantly correlated with SDMT, putamen (r = 0.409, p < 0.001) and thalamus (r = 0.362, p < 0.001) having the strongest effects, whereas for PASAT, the correlation was significant for putamen (r = 0.313, p < 0.01) but not for thalamus. We confirm the significant role of thalamus atrophy in MS-related IPS slowing and find that putamen atrophy is also a significant contributor to this disorder. These DGM structures have independent, significant roles, after controlling for the influence of neocortex atrophy.
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Gânglios da Base/patologia , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/psicologia , Esclerose Múltipla/patologia , Esclerose Múltipla/psicologia , Neocórtex/patologia , Tálamo/patologia , Adulto , Atrofia , Interpretação Estatística de Dados , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/complicações , Testes Neuropsicológicos , Valor Preditivo dos TestesRESUMO
OBJECTIVE: To investigate abnormal phase on susceptibility-weighted imaging (SWI)-filtered phase images indicative of iron content, in subcortical deep-gray matter (SDGM) of multiple sclerosis (MS) patients and healthy controls (HC), and to explore its relationship with MRI outcomes. METHODS: 169 relapsing-remitting (RR) and 64 secondary-progressive (SP) MS patients, and 126 age- and sex-matched HC were imaged on a 3T scanner. Mean phase of the abnormal phase tissue (MP-APT), normal phase tissue volume (NPTV) and normalized volume were determined for total SDGM, caudate, putamen, globus pallidus, thalamus, pulvinar nucleus of thalamus (PVN), hippocampus, amygdala, nucleus accumbens, red nucleus and substantia nigra. 63 HC were used for establishment of normal reference phase values, while additional 63 HC were used for blinded comparisons with MS patients. RESULTS: Increased MP-APT, decreased normalized volume and decreased NPTV were detected in total SDGM, caudate, putamen, globus pallidus, thalamus and PVN in MS patients compared to HC (p<.0004). MS patients also showed decreased volume in hippocampus (<.0001) and decreased NPTV in the hippocampus, amygdala and accumbens (<.0004). SPMS patients had increased MP-APT, decreased volume and decreased NPTV in total SDGM, caudate and amygdala compared to RRMS (p<.005), while individual measure differences were also detected in putamen, thalamus, hippocampus and accumbens (p<.006). RRMS patients showed a significant relationship between increased MP-APT and increased lesion burden and more advanced brain atrophy (p<.004). CONCLUSIONS: Abnormal phase, indicative of higher iron content was significantly increased in MS patients compared to HC, and was related to more severe lesion burden and brain atrophy.
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Encéfalo/patologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento por Ressonância Magnética/métodos , Esclerose Múltipla/patologia , Encéfalo/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Ferro/análise , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE: The associations between vitamin D and MRI measures of brain tissue injury have not been previously investigated in multiple sclerosis (MS). This research evaluates the significance of vitamin D and its active metabolites in brain tissue injury and clinical disability in MS patients. METHODS: The study population consisted of 193 MS patients (152 women and 41 men; mean age 46.1 (SD 8.4) years; disease duration 13.8 (SD 8.4) years). Serum levels of 25-hydroxyvitamin D(3) (25(OH)VD(3)), 25-hydroxyvitamin D(2) (25(OH)VD(2)), 1α, 25-dihydroxyvitamin D(3) (1, 25(OH)(2)VD(3)) and 24(R), 25-dihydroxyvitamin D(3) (24, 25(OH)(2)VD(3)) were measured using a novel capillary liquid-chromatography-mass spectrometry method. Disability was assessed with the Expanded Disability Status Scale (EDSS) and the MS Severity Scale (MSSS). MRI measures included T2 lesion volume (LV), T1-LV and brain parenchymal fraction. The associations between deseasonalised levels of vitamin D metabolites and clinical and MRI measurements were assessed using regression analyses. RESULTS: Lower deseasonalised levels of total 25(OH)VD (p=0.029), 25(OH)VD(3) (p=0.032) and 24, 25(OH)(2)VD(3) (p=0.005) were associated with higher MSSS. Similarly, lower deseasonalised levels of 24, 25(OH)(2)VD(3) (p=0.012) were associated with higher EDSS. Higher values of the 25(OH)VD(3) to 24, 25(OH)(2)VD(3) ratio were associated with higher MSSS (p=0.041) and lower brain parenchymal fraction (p=0.008). CONCLUSIONS: Vitamin D metabolites have protective associations with disability and brain atrophy in MS. In particular, the results indicate strong associations for the 24, 25(OH)(2)VD(3) metabolite, which has not been extensively investigated in MS patients.
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Esclerose Múltipla/sangue , Esclerose Múltipla/patologia , Vitamina D/sangue , 24,25-Di-Hidroxivitamina D 3/sangue , 25-Hidroxivitamina D 2/sangue , Adulto , Calcifediol/sangue , Calcitriol/sangue , Cromatografia Líquida , Avaliação da Deficiência , Progressão da Doença , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Análise de RegressãoRESUMO
Abstract Early in the HIV/AIDS epidemic in the United States, relatively few women were diagnosed with HIV infection and AIDS. Today, the epidemic represents a growing and persistent health threat to women in the United States, especially young women and women of color. In 2005, the leading cause of HIV infection among African American women and Latinas was heterosexual contact. In addressing HIV prevention needs among women, community-level strategies are needed to increase consistent condom use by women and their partners and to change community norms to support safer sex behaviors. The Real AIDS Prevention Project (RAPP) is a community-based HIV prevention intervention for women and their partners. RAPP is based on a community mobilization model that involves a combination of activities, including street outreach, one-on-one discussions called stage-based encounters, role model stories, community networks, and small group activities. The objectives of RAPP are to increase consistent condom use by women and their partners and change community norms associated with perceptions of condom use and high-risk behaviors in an effort to make safer sex practice more acceptable. This paper describes the Centers for Disease Control and Prevention (CDC) Division of HIV/AIDS Prevention (DHAP) effort to nationally diffuse RAPP from March 2003 through May 2007 and lessons learned from that diffusion experience. The paper specifically discusses (1) collaborating and planning with researchers, (2) a diffusion needs assessment that was designed to assess prior implementation experiences among select agencies, (3) developing the intervention package, (4) developing and piloting training for community-based organizations (CBOs), (5) a rollout of national trainings for health departments and community-based organizations interested in implementing RAPP, and (6) ongoing quality assurance activities and the provision of technical assistance and support. RAPP has been proven effective in reducing HIV transmission risk behaviors and improving communication and negotiation skills necessary for African American women and Latinas to reduce their risk for HIV infection and improve their overall health status.
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Participação da Comunidade/métodos , Difusão de Inovações , Infecções por HIV/prevenção & controle , Promoção da Saúde/métodos , Negro ou Afro-Americano , Centers for Disease Control and Prevention, U.S. , Redes Comunitárias , Preservativos , Comportamento Cooperativo , Feminino , Hispânico ou Latino , Humanos , Avaliação das Necessidades , Projetos Piloto , Desenvolvimento de Programas , Estados Unidos , Saúde da MulherRESUMO
The disproportionate rates of HIV/AIDS among African American women in the U.S. signify the ongoing need for targeted HIV prevention interventions. Additionally, building the capacity of service providers to sustain prevention efforts is a major concern. The Centers for Disease Control and Prevention (CDC) conducted a pilot project to disseminate the Sisters Informing Sisters about Topics on AIDS (SISTA), an HIV prevention intervention designed for African American women. The project was to inform the diffusion process and examine the training and technical assistance needs of participating community-based organizations. Results demonstrated a need for extensive pre-planning and skills-building prior to implementation.
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Negro ou Afro-Americano/estatística & dados numéricos , Relações Comunidade-Instituição , Infecções por HIV/prevenção & controle , Promoção da Saúde/estatística & dados numéricos , Educação Sexual/estatística & dados numéricos , Saúde da Mulher/etnologia , Adulto , Redes Comunitárias , Feminino , Infecções por HIV/etnologia , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Pessoa de Meia-Idade , Inovação Organizacional , Projetos Piloto , Avaliação de Programas e Projetos de Saúde , Comportamento de Redução do Risco , Estados Unidos/epidemiologiaRESUMO
Although race and gender are not indicators for HIV/AIDS, both have disproportionately impacted African American women. African American women represent 13% of the U.S. female population and 67% of the AIDS cases among women (Fitzpatrick, The U.S. HIV/AIDS Epidemic in Women and Adolescent Females, HIV Prevention Conference, Atlanta, GA, 2005). The statistics underscore the need for targeted interventions that employ culturally relevant activities to enhance self-esteem and communication skills while encouraging positive behavior change. Factors facilitating intervention effectiveness include culturally relevant components such as cultural practices, beliefs, values, norms, and ideologies (Janz et al., "Evaluation of 37 AIDS Projects," Health Education Quarterly, 23(1), 80-97, 1996). HIV prevention programs targeting African American women should incorporate an approach that includes ethnic heritage as ameans to instill pride, therebymotivating positive behavior change and empowering women. Afrocentric approaches incorporate philosophies relevant to people of African descent and may be spiritually based. Coupling culturally relevant HIV prevention interventions with a culturally relevant diffusion strategy may enhance community receptiveness. The SISTA intervention (DiClemente & Wingood, "A Randomized Controlled Trial of an HIV Sexual Risk Reduction Intervention for Young African-American Women," Journal of the American Medical Association, 274(16), 1271-1276, 1995) incorporates both culturally and gender-relevant activities to empower African American women to make healthy life choices. The article presents the strategy used to nationally diffuse SISTA, which incorporated Afrocentric components within implementation delivery. Lessons learned demonstrate the significance of integrating additional Afrocentric and gender-relevant material to an existing intervention for African American women.
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Negro ou Afro-Americano , Difusão de Inovações , Infecções por HIV/prevenção & controle , Promoção da Saúde/organização & administração , Adolescente , Redes Comunitárias , Diversidade Cultural , Feminino , Humanos , Estudos de Casos Organizacionais , Estados UnidosRESUMO
Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.
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alfa-Globulinas/isolamento & purificação , Plasma/química , Proteômica , alfa-Globulinas/análise , Sequência de Aminoácidos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Inibidores de Serina Proteinase/sangue , Inibidores de Serina Proteinase/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Inibidor da Tripsina de Soja de Kunitz/sangue , Inibidor da Tripsina de Soja de Kunitz/isolamento & purificaçãoRESUMO
For proteomic analysis, plasma membranes of rat hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized according to the previously developed method [D. Josic, K. Zeilinger, Methods Enzymol. 271 (1996) 113-134]. If the Triton X100 insoluble pellet is subsequently extracted, several proteins can be solubilized. These proteins can be classified in two groups according to their molecular size. The proteins with apparent molecular weights in SDS-PAGE between 70 and 75 kDa belong to the first group. Smaller proteins, with apparent molecular weights between 30 and 45 kDa, are members of the second group. The main protein of higher molecular weight was also found in the Triton X100 insoluble extract from normal rat liver plasma membranes. This protein was identified as Annexin A6. The proteins from the second group are practically absent in the Triton X100 insoluble extract from rat liver. These proteins are present in relatively high concentrations in plasma membranes of Morris hepatoma 7777. Both groups of detergent-insoluble proteins from Morris hepatoma 7777 were further analyzed with SELDI-TOF and LC electrospray ionization mass spectrometry. From the first group, Annexin A6, together with two other integral plasma membrane proteins, was identified. In the second group of proteins with apparent molecular weights between 30 and 45kDa, further members of the annexin family, Annexins A1, A2, A4, A5 and A7 were identified. The possible role of these low molecular size annexins as potential cancer biomarkers is discussed.
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Anexinas/isolamento & purificação , Membrana Celular/química , Neoplasias Hepáticas Experimentais/química , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais/análise , Cálcio/farmacologia , Cromatografia Líquida , Ácido Egtázico/farmacologia , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Octoxinol , Ratos , Solubilidade , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.